PROJECT SUMMARY/ABSTRACT The mechanisms of pathogen sensing and immune effector induction in intestinal epithelial cells are not completely understood. Disruption in the mechanisms of pathogen sensing and immune homeostasis in intestinal epithelial cells can lead to dysbiosis and inflammation, as well as susceptibility to bacterial infection. Key insights into intestinal epithelial cell immunity and host-pathogen interactions have been made using the nematode C. elegans. Nematodes mount innate immune defenses against bacterial infection via conserved immune pathways, but the mechanisms of pathogen detection are unknown in this organism. In nematodes, the family of nuclear hormone receptors (NHRs) has dramatically expanded compared to other metazoans. NHRs are ligand-gated transcription factors that sense endogenous and exogenous signals to induce adaptive transcriptional responses. The C. elegans genome encodes 274 NHRs, of which 260 are homologs of human HNF4?. HNF4? is a key NHR involved in inflammatory bowel disease, though the mechanism through which HNF4? mediates inflammatory bowel disease in humans is unknown. The central hypothesis of this proposal is that C. elegans HNF4? homologs are an ancient family of pathogen sensors whose evolutionary expansion in C. elegans was driven by their function in detecting diverse pathogens. The following key findings support this hypothesis: (i) The nuclear hormone receptor, NHR-86/HNF4?, senses the cellular environment and activates C. elegans intestinal immune defenses; (ii) NHR-86/HNF4? is required for pathogen resistance and immune response towards the gram positive human pathogen E. faecalis; and (iii) A different C. elegans HNF4? homolog is required for pathogen defense and immune effector regulation against the gram negative pathogen P. aeruginosa. In this proposal, Aim 1 will define the role of C. elegans NHR-86/HNF4? in pathogen detection and immune effector induction during E. faecalis infection using a combination of transcriptomics, ChIP- sequencing, tissue-specific rescue and genetic epistasis. Aim 2 will characterize the function of a separate C. elegans HNF4? homolog in pathogen sensing during P. aeruginosa infection. The approach includes: transcriptomics, global NHR binding site identification, tissue specific rescue, and P. aeruginosa genetics. Collectively, these studies will characterize a fundamentally new paradigm of immune activation, which will solve a major conundrum of how pathogens are sensed in C. elegans. These findings will also establish NHRs as evolutionarily ancient pathogen sensors. Ultimately, the expectation is that detailed dissection of this mechanism will shed light on the role of HNF4? in mammalian pathogen sensing and inflammatory bowel disease.